Purpose of the Study: This study was conducted to detect and characterize the genes encoding extended spectrum β-lactamases and associated β-lactamases (carbapenemases and Ambler Class C β-lactamases).Patients and Methods: In 2011, out of the 65 non-duplicative Klebsiellapneumoniae collected from blood culture at Charles Nicolle hospital of Tunisia, 36 were resistant to 3rd generation cephalosporin.Results: All strains showed a double disk synergy test positive. They were mainly isolated in intensive care unit (31%). They were frequently resistant to most antibiotics tested, except colistin and tigecyclin. Five isolates (13%) showed reduced susceptibility to carbapenems. blaCTX-M-15 was harbored by 35 strains and blaSHV-12 by one. blaCTX-M-15 were associated with blaTEM-1 (n=21), blaOXA-48 and blaCMY-2 (n=1) and blaOXA-48and blaTEM-1 (n=4). The conjugation wassuccessfulfor4/5 strains (3 harboring blaCTX-M-15 and one blaSHV-12). The plasmids carrying the blaCTX-M-15 were assigned to IncN or IncL/M only for 2 strains. The remaining blaCTX-M-15-carrying plasmid was negative for all of the replicons tested as well as the blaSHV-12-carrying plasmid.Conclusion: Our results confirm the spread of CTX-M-15 in our institution. To our knowledge, this is the first report of K. pneumoniae coproducing CTX-M-15, CMY-2 and OXA-48. The implementation of preventive measures against the spread of these multiresistant bacteria is needed.

Since their description in the mid-1980s, extended spectrum β-lactamase (ESBL)-producing organisms have become recognized as a worldwide problem.[1] Although ESBLs have been detected in a wide variety of Gram-negative bacteria, Klebsiellapneumoniae has been found to be the most common species to produce ESBLs.[2] Because ESBL- producing organisms are frequently multidrug resistant, therapeutic options for these infections are severely limited and treatment can be challenging. Also, there have been many reports of outbreaks caused by these organisms, and it has been demonstrated that ESBL production by infecting organisms adversely affects the clinical outcome with a significant morbidity and mortality.[3],[4],[5],[6]

In Tunisia, epidemiological data of the spread of ESBL-producing K. pneumoniaestrains showed a rapid diffusion since their first description in 1984.[7] In fact, their prevalence varies from 27.9% in 1999 to 51.5% in 2007[8] with a high incidence 87.5% in Mongi Slim Hospital followed by 19°/Ω[4] in Children's Hospital.

Among the 65 non-duplicate K. pneumoniae isolated from blood cultures of 65 patients hospitalized at Charles Nicolle Hospital of Tunis from January to December 2011, 36 (55.3%) were resistant to 3rd GC. They were collected mainly from intensive care unit (31%), neonatology (26%) and surgery (23%) [Table 1].

Antimicrobial susceptibility testing was determined by the disk-diffusion method on Mueller-Hinton (MH) agar plates (Bio-Rad, Marnes-la-Coquette, France) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.[9] ESBL production was detected by the double disk synergy test (DDST) with or without cloxacillin as described previously.[10] Isolates with reduced susceptibility to carbapenems: imipenem (diameter zone ≤ 22 mm) and/or ertapenem (diameter zone ≤ 18 mm) were subjected to the modified Hodge screening test (MHT) for diffusible carbapenemase detection as previously described.[11] The minimal inhibition concentrations (MICs) of ceftazidime, ceftazidime-clavulanic acid, cefotaxime, cefoxitine and cefepime were determined by agar dilution technique. For imipenem, ertapenem and meropenem, MICs were determined by E-test (Bio-Mérieux SA, Marcy l'étoile, France). Results were interpreted according to the CLSI guidelines.[9]

Detection of β-lactamase-encoding Genes

K. pneumoniae isolates were screened by PCR for the following β-lactamases encoding genes: blaCTX-M phylogenetic lineage groups 1, 2 and 9,[12],[13],[14]blaTEM, blaSHV, blaCIT'blaACCblaEBCblaMOXblaFOX, blaDHA, blaLAT, blaACT, blaMIR,[15]blaOXA-48 and blaKpC[16] as described previously. The PCR products were purified with a Purification Kit (BioMatik) and sequenced on an ABI PRISM 310 DNA Sequencer (Applied Biosystems). The nucleotide sequences were analyzed with the BLAST program (http://www.ncbi.nlm. nih.gov/BLAST/).

β-Lactam Resistance Transfer Assays

Conjugation assays were performed on 5 isolates selected on the basis of their susceptibility to rifampin. It was carried out in brain heart broth (Bio-Rad), with E. coli J53-2 as the recipient. Transconjugants were selected on Mueller Hinton agar plates containing rifampicin (500 mg/L) and ticarcillin (125 mg/L). If not successful at the first attempt, mating experiments were repeated up to 3 times. Transconjugant's were submitted to bacterial identification with the API-20E system (Bio-Mérieux SA, Marcy l'étoile, France), antibiotic susceptibility testing and PCR amplification of the bla genes mentioned above.

All isolates showed a positive DDST. They showed high level of resistance to cefotaxime (MIC50= 512 μg/mL), ceftazidime (MIC50=128 μg/mL) and cefepóme (MIC50=128 μg/mL). MICs of ceftazidime decreased when clavulanic acid is added. However, 1 strain showed high level of resistance to cefoxitin (MIC=>512 μg/mL) [Table 1]. Five strains were resistant to ertapenem and showed a positive MHT. Three of them were resistant to imipinem and/or meropenem [Table 1].

The results of PCR and sequence analysis are summarized in [Table 1]. The blaSHV1 gene was detected in all K. pneumoniae strains. ESBL genes were detected in all strains, blaCTX-M-15 in 35 strains and blaSHV-12 in one. Moreover, 28 isolates harbored blaTEM-1. Ertapenem resistant strains carried blaOXA-48. The blaCMY-2 genewas detected in one strain that also coproduce 3β-lactamases genes (blaCTX-M-15, blaOXA-48 and blaTEM-1).

Plasmid Replicon Type Determination

For 2 strains, the plasmids carrying blaCTX-M-15 were assigned to the IncN and IncL/M replicon type. However, the remaining blaCTX-M-15-carrying plasmid was negative for all of the replicons tested, as well as the blaSHV-12-carrying plasmid [Table 1].

ESBL Transfer

Theconjugationwassuccessfulfor4/5 strains (3 harboring blaCTX-M-15 and one blaSHV-12). We also observed co-transfer, following conjugation, of resistance to other antibiotics, such as i.e. gentamicin (n=1), tobramycin (n=2), netilmicin (n=1), minocycline (n=4) and chloramphenicol (n=2).

Production of ESBLs was detected in all transconjugants by the DDST. The transfer of ESBL genes was confirmed in all transconjugants; blaCTX-M-15 and blaSHV-12 were detected in 3 and 1 transconjugants, respectively. Only one of the 4 transconjugants was typeable for the incompatibility groups (IncL/M) [Table 2].

Table 2: Resistance profils and β-lactamases genes of donor strains and their transconjugants

ESBL producing K. pneumoniae is one of the most important pathogens responsible for life threatening infections.[4],[18],[19] In the present study, frequency of ESBL producing K. pneumoniae causing bacteremia is 47% which is lower than 87.5% reported in a Tunisian pediatric intensive care unit.[4] A study conducted in India reported 56% ESBL K. pneumoniae causing septicemiaamongneonates.[18]

In most European countries, Latin America, and East Asia, CTX-M variants have displaced TEM and SHV enzymesas the predominant β-lactamases produced by Gram-negative bacteria such as K. pneumoniae.[20] Our work shows that CTX-M-15 is the most common ESBL. CTX-M-15, first described in India in 1999,[21] has spread over the world, and seems to be the most common ESBL type.[22],[23] In Tunisia, dissemination of CTX-M-15 has been reported in K. pneumoniae, E. coli and Enterobacter cloacae strains.[2],[5],[24],[25]blaCTX-M-15 gene is generally found on large conjugative plasmids and is located downstream of an ISEcpl insertion sequence which explains its remarkable transmission success.[26] The large use of 3rdGC in clinical practice has significantly contributed to their selection.[27]

Although SHV-12 was found in one isolate of our collection, previous Tunisian studies showed that this enzyme was associated with K. pneumoniaeinvolved in nosocomial outbreaks.[2],[3],[20],[28],[29] Worldwide, the distribution of SHV- ESBLs showed that SHV-2, SHV-4 and SHV-5 enzymes are the most prevalent in the United States and Europe.[30]

The ESBL-producing organisms additionally exhibited co-resistance against multiple antimicrobials from other classes, such as tetracycline, aminoglycosides and fluoroquinolones. This complexity in antimicrobials resistance combinations limits suitable drug of choice for antimicrobial therapy, leaving carbapenems the last options for treatment in some cases. However, the emergence of plasmidiccarbapenemases producers clearly compounds potential treatment options.[14] Carbapenem resistance was found in 5 of our strains. All harbored OXA-48. This class D carbapenemasewas first identified in K. pneumoniae from Turkey in 2003[16] and then extensively spread in other countries[31] and species. In Tunisia, recent studies reported the spread of OXA-48 producing Enterobacteriaceae.[32],[33]

blaCMY-2 is the most frequently encountered plasmid encoded AmpC-type β-lactamase gene found in E. coli worldwide.[34],[35],[36] the first AmpC type β-lactamase to be identified in Tunisia was in 1996 in a P. mirabilie isolate, and was characterized as a CMY-4 enzyme.[37] In Tunisia, CMY-2 has already been detected in food samples[34] and fecal flora of healthy chickens and pets[38] but never in clinical isolates. In our study, blaCMY-2 was detected in one strain, which co-produced 3 plasmidicβ-lactamases (CTX-M-15, OXA-48 and TEM-1). The coexistence of several enzymes in the same strain is noteworthy and has been previously described in K. pneumoniae in Tunisia (VIM-4, CTX-M-15 and CMY-4).[39]

Identification and classification of plasmids harbouring β-lactamases is helpful to analyze their distribution in nature and their relationship to host cells and to discover their evolutionry origins.[40] In our study, incompatibility groups of blaCTX-M-15 carrying plasmids were determined only in 2 strains (IncN and IncL/M). However, other studies reported that some blaCTX-M-15 and blaSHV-12-carrying plasmids were negative for all of the replicons tested,[2],[28] as well as described in our study. Further research is necessary to provide more information about our plasmids.

In conclusion, our study reports a widespread diffusion of CTX-M-15 with the emergence of OXA-48 in K. pneumoniae in Tunisia which seriously limits therapeutic options in cases of clinical infections with these bacteria. In addition, coexistence of ESBLs production with cephalosporinase or carbapenemase is a serious public health problem and requires continuous surveillance, monitoring and revision of the antibiotic use policies.

Acknowledgments

This work was funded by Grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology.

Disclosure Statement

All authors disclose no commercial associations that might create a conflict of interest in connection with this study.